Anti-tumor T cell as well as preparation method and anti-tumor drug thereof

An anti-tumor drug, tumor cell technology, applied in anti-tumor drugs, animal cells, drug combinations, etc., can solve the problem of no malignant brain tumor clinical data and other problems

Inactive Publication Date: 2015-05-20
深圳市中美康士生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the small size of resected tumor samples and the limited availability of TILs, t

Method used

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  • Anti-tumor T cell as well as preparation method and anti-tumor drug thereof
  • Anti-tumor T cell as well as preparation method and anti-tumor drug thereof
  • Anti-tumor T cell as well as preparation method and anti-tumor drug thereof

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preparation example Construction

[0056] The second aspect of the present invention is to provide a method for preparing anti-tumor T cells as described above, comprising the following steps:

[0057] Step 1. Preparation of total tumor RNA: synthesize tumor total RNA from cultured tumor cells in vitro;

[0058] Step 2. Preparation of mature tumor total RNA-DC: the tumor total RNA obtained in step 1 and the isolated dendritic cell DC were introduced into the DC by electrotransduction, and then cultured overnight in the culture medium to obtain mature tumor total RNA-DC. RNA-DC;

[0059] Step 3. Co-culture the mature tumor total RNA-DC obtained in step 2 with T cells to obtain the anti-tumor T cells according to claim 1.

[0060] In the above preparation method, preferably, the tumor cell line in step 1 is from human or mouse tumor cells, more preferably from but not limited to the highly invasive mouse astrocytoma cell line KR158 and melanoma cell line B16F10OVA.

[0061] In the above preparation method, prefe...

Embodiment

[0072] 1. Materials and methods:

[0073] 1. Cell culture and ttRNA preparation from mouse tumor cell lines:

[0074] Mouse cytokines IL-4, IL-7, IL-12, IL-15, IL-21, GM-CSF and CD40L were purchased from PEPROTECH (Los Angeles, New Jersey), and IL-2 (Aldesleukin) was purchased from PROLEUKIN ( San Diego, California). Mouse T cells were cultured in RPMI containing 5% FBS supplemented with 100 U / ml penicillin / streptomycin, 1 mM L-glutamine, 55 uM β-ME, 1 mM sodium pyruvate, 0.1 mM NEAA, 10 mM HEPES (Invitrogen Corporation). In this example, the highly invasive mouse astrocytoma model and melanoma IC tumor model are preferred, that is, from NF1 - / - :p53 - / - The astrocytoma cell line KR158 in C57BL / 6-mice, and the melanoma cell line B16F10OVA from mice cultured in DMEM with 10% FBS. All cells were cultured at 37°C in a 5% CO2 humidified incubator. Lipopolysaccharide (LPS) was purchased from Sigma (St. Louis, MO). The total tumor RNA (total tumor RNA, ttRNA) from KR158 and B...

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Abstract

The invention provides a method for preparing an anti-tumor T cell in vitro through DC (dendritic cell, namely antigen presenting cell), wherein the anti-tumor T cell is a tumor total RNA-loaded DC amplification and enrichment anti-tumor T cell. The invention also provides a preparation method of the anti-tumor T cell and an anti-tumor drug or inoculation vaccine taking the anti-tumor T cell as an active ingredient. According to the invention, a specific anti-tumor T cell is amplified in vitro by taking the tumor total RNA-loaded DC as a platform, a small amount of tumor total RNA is amplified to an enough amount in vitro so as to realize clinical application, and the in-vivo anti-tumor effect in an IC (intracranial) tumor-bearing model is evaluated, including how the mature state of DC and the cell factor combination adjust the differentiation of the anti-tumor T cell as well as the synergistic anti-tumor effect thereof in a preclinical experiment model.

Description

technical field [0001] The invention relates to an anti-tumor T cell and a preparation method thereof, as well as an anti-tumor drug or vaccination which uses the anti-tumor T cell as an active ingredient. Background technique [0002] The direct use of in vitro expanded anti-tumor T cells is based on two facts: 1) patients with malignant brain tumors have strong systemic immunosuppression, which seriously hinders the effect of anti-tumor vaccination methods; Compared with adoptive cell transfer (ACT) using in vitro expanded anti-tumor T cells including tumor infiltrated lymphocytes (TIL) or genetically engineered peripheral blood lymphocytes (PBL) Can more effectively eradicate residual tumor cells in melanoma patients (Dudley ME, Yang JC, Sherry R, ​​Hughes MS, Royal R, et al. (2008)Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation Preparative regimens. J Clin Oncol 26:5233-5239; Morgan RA, Dudley ME, Wunderl...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/13A61K39/00A61P35/00
Inventor 杨世成
Owner 深圳市中美康士生物科技有限公司
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